Publications by authors named "Alessandro Aliverti"

42 Publications

Ferredoxin-NADP Reductase-Catalyzed Redox Cycling of Plasmodione Generates Both Predicted Key Drug Metabolites: Implication for Antimalarial Drug Development.

ACS Infect Dis 2021 Apr 15. Epub 2021 Apr 15.

Université de Strasbourg-CNRS-UHA UMR7042, Laboratoire d'Innovation Moléculaire et Applications (LIMA), Team Bio(IN)organic and Medicinal Chemistry, European School of Chemistry, Polymers and Materials (ECPM), 25 Rue Becquerel, F-67087 Strasbourg, France.

Plasmodione (PD) is a potent antimalarial redox-active 3-benzyl-menadione acting at low nanomolar range concentrations on different malaria parasite stages. The specific bioactivation of PD was proposed to occur via a cascade of redox reactions starting from one-electron reduction and then benzylic oxidation, leading to the generation of several key metabolites including corresponding benzylic alcohol (PD-bzol, for PD benzhydrol) and 3-benzoylmenadione (PDO, for PD oxide). In this study, we showed that the benzylic oxidation of PD is closely related to the formation of a benzylic semiquinone radical, which can be produced under two conditions: UV photoirradiation or catalysis by apicoplast ferredoxin-NADP reductase (FNR) redox cycling in the presence of oxygen and the parent PD. Electrochemical properties of both PD metabolites were investigated in DMSO and in water. The single-electron reduction potential values of PD, PD-bzol, PDO, and a series of 3-benzoylmenadiones were determined according to ascorbate oxidation kinetics. These compounds possess enhanced reactivity toward FNR as compared with model quinones. Optimal conditions were set up to obtain the best conversion of the starting PD to the corresponding metabolites. UV irradiation of PD in isopropanol under positive oxygen pressure led to an isolated yield of 31% PDO through the transient semiquinone species formed in a cascade of reactions. In the presence of FNR, PDO and PD-bzol could be observed during long lasting redox cycling of PD continuously fueled by NADPH regenerated by an enzymatic system. Finally, we observed and quantified the effect of PD on the production of oxidative stress in the apicoplast of transgenic 3D7 parasites by using the described genetically encoded glutathione redox sensor hGrx1-roGFP2 methodology. The observed fast reactive oxygen species (ROS) pulse released in the apicoplast is proposed to be mediated by PD redox cycling catalyzed by FNR.
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http://dx.doi.org/10.1021/acsinfecdis.1c00054DOI Listing
April 2021

Ligand Binding in Allosteric Flavoproteins: Part 2. Quantitative Analysis of the Redox-Dependent Interaction of the Apoptosis-Inducing Factor (AIF) with Its Protein Partner.

Methods Mol Biol 2021 ;2280:189-198

Department of Biosciences, University of Milan, Milan, Italy.

To perform their action usually flavoproteins interact transiently with a variety of molecular partners, whose binding is reciprocally affected and often controlled by the redox state of the bound flavin cofactor. As a case study, here we describe an approach for the quantitative characterization of the redox-controlled interaction of the mammalian apoptosis inducing factor (AIF) with one of its known protein partners, namely, the mitochondrial coiled-coil-helix-coiled-coil-helix domain-containing protein 4 (CHCHD4). In particular, we report a protocol for the titration of the flavoprotein in both in its oxidized and reduced states with CHCHD4, using an implementation of the MicroScale Thermophoresis (MST) technique.
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http://dx.doi.org/10.1007/978-1-0716-1286-6_12DOI Listing
January 2021

Ligand Binding in Allosteric Flavoproteins: Part 1. Quantitative Analysis of the Interaction with NAD of the Apoptosis Inducing Factor (AIF) Harboring FAD in the Reduced State.

Methods Mol Biol 2021 ;2280:179-187

Department of Biosciences, University of Milan, Milan, Italy.

To perform their action, flavoproteins usually interact with a variety of low molecular weight partners, including electron transporters, yielding transient complexes whose tightness is often controlled by the redox state of the bound flavin cofactor. As a case study, here we describe the quantitative analysis of the redox-dependent interaction of the mammalian apoptosis inducing factor (AIF) with its NAD ligand. In particular, we report a protocol for the spectrophotometric titration of AIF in its reduced state under anaerobic conditions with NAD, in order to determine the dissociation constant of the resulting complex.
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http://dx.doi.org/10.1007/978-1-0716-1286-6_11DOI Listing
January 2021

Reactions of Ferredoxin:NADP Oxidoreductase with Redox Cycling Xenobiotics: A Mechanistic Study.

Int J Mol Sci 2020 May 2;21(9). Epub 2020 May 2.

Department of Xenobiotics Biochemistry, Institute of Biochemistry of Vilnius University, Saulėtekio 7, LT-10257 Vilnius, Lithuania.

Ferredoxin:NADP oxidoreductase from (FNR) catalyzes the NADPH-dependent reduction of ferredoxin (Fd), which provides redox equivalents for the biosynthesis of isoprenoids and fatty acids in the apicoplast. Like other flavin-dependent electrontransferases, FNR is a potential source of free radicals of quinones and other redox cycling compounds. We report here a kinetic study of the reduction of quinones, nitroaromatic compounds and aromatic oxides by FNR We show that all these groups of compounds are reduced in a single-electron pathway, their reactivity increasing with the increase in their single-electron reduction midpoint potential (). The reactivity of nitroaromatics is lower than that of quinones and aromatic -oxides, which is in line with the differences in their electron self-exchange rate constants. Quinone reduction proceeds via a ping-pong mechanism. During the reoxidation of reduced FAD by quinones, the oxidation of FADH to FAD is the possible rate-limiting step. The calculated electron transfer distances in the reaction of FNR with various electron acceptors are similar to those of FNR, thus demonstrating their similar "intrinsic" reactivity. Ferredoxin stimulated quinone- and nitro-reductase reactions of FNR, evidently providing an additional reduction pathway via reduced Fd. Based on the available data, FNR and possibly Fd may play a central role in the reductive activation of quinones, nitroaromatics and aromatic oxides in contributing to their antiplasmodial action.
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http://dx.doi.org/10.3390/ijms21093234DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7247349PMC
May 2020

Antiplasmodial Activity of Nitroaromatic Compounds: Correlation with Their Reduction Potential and Inhibitory Action on Glutathione Reductase.

Molecules 2019 Dec 10;24(24). Epub 2019 Dec 10.

Department of Xenobiotics Biochemistry, Institute of Biochemistry of Vilnius University, Saulėtekio 7, LT-10257 Vilnius, Lithuania.

With the aim to clarify the mechanism(s) of action of nitroaromatic compounds against the malaria parasite , we examined the single-electron reduction by ferredoxin:NADP oxidoreductase (FNR) of a series of nitrofurans and nitrobenzenes ( = 23), and their ability to inhibit glutathione reductase (GR). The reactivity of nitroaromatics in FNR-catalyzed reactions increased with their single-electron reduction midpoint potential (). Nitroaromatic compounds acted as non- or uncompetitive inhibitors towards GR with respect to NADPH and glutathione substrates. Using multiparameter regression analysis, we found that the in vitro activity of these compounds against strain FcB1 increased with their values, octanol/water distribution coefficients at pH 7.0 (log ), and their activity as GR inhibitors. Our data demonstrate that both factors, the ease of reductive activation and the inhibition of GR, are important in the antiplasmodial in vitro activity of nitroaromatics. To the best of our knowledge, this is the first quantitative demonstration of this kind of relationship. No correlation between antiplasmodial activity and ability to inhibit human erythrocyte GR was detected in tested nitroaromatics. Our data suggest that the efficacy of prooxidant antiparasitic agents may be achieved through their combined action, namely inhibition of antioxidant NADPH:disulfide reductases, and the rapid reduction by single-electron transferring dehydrogenases-electrontransferases.
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http://dx.doi.org/10.3390/molecules24244509DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6943496PMC
December 2019

Cellulose production is coupled to sensing of the pyrimidine biosynthetic pathway via c-di-GMP production by the DgcQ protein of Escherichia coli.

Environ Microbiol 2017 11 21;19(11):4551-4563. Epub 2017 Sep 21.

Department of Biosciences, Università degli Studi di Milano, Milan, Italy.

Production of cellulose, a stress response-mediated process in enterobacteria, is modulated in Escherichia coli by the activity of the two pyrimidine nucleotide biosynthetic pathways, namely, the de novo biosynthetic pathway and the salvage pathway, which relies on the environmental availability of pyrimidine nitrogenous bases. We had previously reported that prevalence of the salvage over the de novo pathway triggers cellulose production via synthesis of the second messenger c-di-GMP by the DgcQ (YedQ) diguanylate cyclase. In this work, we show that DgcQ enzymatic activity is enhanced by UTP, whilst being inhibited by N-carbamoyl-aspartate, an intermediate of the de novo pathway. Thus, direct allosteric control by these ligands allows full DgcQ activity exclusively in cells actively synthesizing pyrimidine nucleotides via the salvage pathway. Inhibition of DgcQ activity by N-carbamoyl-aspartate appears to be favoured by protein-protein interaction between DgcQ and PyrB, a subunit of aspartate transcarbamylase, which synthesizes N-carbamoyl-aspartate. Our results suggest that availability of pyrimidine bases might be sensed, somehow paradoxically, as an environmental stress by E. coli. We hypothesize that this link might have evolved since stress events, leading to extensive DNA/RNA degradation or lysis of neighbouring cells, can result in increased pyrimidine concentrations and activation of the salvage pathway.
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http://dx.doi.org/10.1111/1462-2920.13918DOI Listing
November 2017

High-resolution studies of hydride transfer in the ferredoxin:NADP reductase superfamily.

FEBS J 2017 10 29;284(19):3302-3319. Epub 2017 Aug 29.

Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR, USA.

Ferredoxin: NADP reductase (FNR) is an FAD-containing enzyme best known for catalysing the transfer of electrons from ferredoxin (Fd) to NADP to make NADPH during photosynthesis. It is also the prototype for a broad enzyme superfamily, including the NADPH oxidases (NOXs) that all catalyse similar FAD-enabled electron transfers between NAD(P)H and one-electron carriers. Here, we define further mechanistic details of the NAD(P)H ⇌ FAD hydride-transfer step of the reaction based on spectroscopic studies and high-resolution (~ 1.5 Å) crystallographic views of the nicotinamide-flavin interaction in crystals of corn root FNR Tyr316Ser and Tyr316Ala variants soaked with either nicotinamide, NADP , or NADPH. The spectra obtained from FNR crystal complexes match those seen in solution and the complexes reveal active site packing interactions and patterns of covalent distortion of the FAD that imply significant active site compression that would favour catalysis. Furthermore, anisotropic B-factors show that the mobility of the C4 atom of the nicotinamide in the FNR:NADP complex has a directionality matching that expected for boat-like excursions of the nicotinamide ring thought to enhance hydride transfer. Arguments are made for the relevance of this binding mode to catalysis, and specific consideration is given to how the results extrapolate to provide insight to structure-function relations for the membrane-bound NOX enzymes for which little structural information has been available.

Databases: Structural data are available in the PDB database under the accession numbers 3LO8 (wild-type), 5VW4 [Y316S:nicotinamide (P3 21)], 5VW9 [Y316S:nicotinamide (P3 21)], 5VW3 [Y316S:NADP (P3 21)], 5VW8 [Y316S:NADP (P3 21)], 5VW2 [Y316S:NADPH (P3 21)], 5VW5 [Y316A:nicotinamide (P3 21)], 5VW6 [Y316A:NADP (P3 21)], 5VW7 [Y316A:NADPH (P3 21)], 5VWA [Y316F (P3 21)], and 5VWB [Y316F:NADP (P3 21)]. Enzyme Commission number: ferredoxin:NADP reductase - E C1.18.1.2.
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http://dx.doi.org/10.1111/febs.14190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5626627PMC
October 2017

Structural bases of the altered catalytic properties of a pathogenic variant of apoptosis inducing factor.

Biochem Biophys Res Commun 2017 08 27;490(3):1011-1017. Epub 2017 Jun 27.

Biophysics Institute, National Research Council c/o Department of Biosciences, Università degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy; Department of Biosciences, Università degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy. Electronic address:

The apoptosis-inducing factor (AIF) is a FAD-containing protein playing critical roles in caspase-independent apoptosis and mitochondrial respiratory chain biogenesis and maintenance. While its lethal role is well known, the details of its mitochondrial function remain elusive. So far, nineteen allelic variants of AIF have been associated to human diseases, mainly affecting the nervous system. A strict correlation is emerging between the degree of impairment of its ability to stabilize the charge-transfer (CT) complex between FAD and NAD and the severity of the resulting pathology. Recently, we demonstrated that the G307E replacement in murine AIF (equivalent to the pathogenic G308E in the human protein) dramatically decreases the rate of CT complex formation through the destabilization of the flavoprotein interaction with NAD(H). To provide further insights into the structural bases of its altered functional properties, here we report the first crystal structure of an AIF pathogenic mutant variant in complex with NAD (murine AIF-G307E) in comparison with its oxidized form. With respect to wild type AIF, the mutation leads to an altered positioning of NAD adenylate moiety, which slows down CT complex formation. Moreover, the altered balance between the binding of the adenine/nicotinamide portions of the coenzyme determines a large drop in AIF-G307E ability to discriminate between NADH and NADPH.
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http://dx.doi.org/10.1016/j.bbrc.2017.06.156DOI Listing
August 2017

Recovery of monosaccharides from lignocellulosic hydrolysates by ion exclusion chromatography.

J Chromatogr A 2017 May 11;1496:25-36. Epub 2017 Mar 11.

Institute for Chemical and Bioengineering, ETH Zurich, Vladimir-Prelog-Weg 1-5/10, 8093 Zurich, Switzerland.

The production of sugars from lignocellulosic biomass is the key to a sustainable, renewable chemical industry. Glucose, xylose and other monosaccharides can be easily produced by hydrolyzing cellulose and hemicellulose, the primary polysaccharides in biomass. However, the hydrolysis of biomass generates byproducts that, together with the mineral acid normally added in the hydrolysis step, have to be removed before the downstream conversion processes. In this work, the recovery of monosaccharides from lignocellulosic hydrolysates by means of Ion Exclusion Chromatography (IEC) has been studied. The analyzed process relies on new pretreatment and hydrolysis steps, involving the neutralization of the hydrolysate with sodium hydroxide. The adsorption behavior of the main components involved in the separation has been experimentally investigated. Pulse tests at the high loading encountered in preparative conditions have been performed for a selected group of model components found in the hydrolysates. For all the electrolytes, the retention volume fraction was always between the interparticle porosity and the total column porosity, confirming that ion exclusion was the dominant retention mechanism. On the other hand, sugars eluted before the total column porosity, indicating partial steric exclusion from the resin pores. This observation was then confirmed by size-exclusion experiments with polyethylene glycol standards, from which the distribution coefficient of the studied sugars has been determined. The comparison between the elution profiles of the same sugars in pure form and as a mixture present in the hydrolysate showed differences in both peak shape and retention times. Therefore, an investigation of the influence of the main electrolytes contained in the hydrolysates on sugars adsorption has been performed through the pulse on a plateau method. The electrolytes were found to enhance the sugars retention by promoting their adsorption onto the resin. However, this effect was not sufficient to explain the observed differences, which were effectively explained in terms of viscous fingering, due to the high viscosity differences between the eluent and the sample. A previously developed model for IEC has been updated to take into account all the observed phenomena and applied to simulate the experimental results. The proposed model was in good agreement with the batch-column elution profiles both for the pure components and for the actual hydrolysate, allowing a quantitative description of the separation.
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http://dx.doi.org/10.1016/j.chroma.2017.03.016DOI Listing
May 2017

Cold Denaturation of the HIV-1 Protease Monomer.

Biochemistry 2017 02 13;56(8):1029-1032. Epub 2017 Feb 13.

Structural Biology and NMR Laboratory (SBiNlab), Department of Biology, University of Copenhagen , Ole Maaloees Vej 5, DK-2200 Copenhagen N, Denmark.

The human immunodeficiency virus-1 (HIV-1) protease is a complex protein that in its active form adopts a homodimer dominated by β-sheet structures. We have discovered a cold-denatured state of the monomeric subunit of HIV-1 protease that is populated above 0 °C and therefore directly accessible to various spectroscopic approaches. Using nuclear magnetic resonance secondary chemical shifts, temperature coefficients, and protein dynamics, we suggest that the cold-denatured state populates a compact wet globule containing transient non-native-like α-helical elements. From the linearity of the temperature coefficients and the hydrodynamic radii, we propose that the overall architecture of the cold-denatured state is maintained over the temperature range studied.
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http://dx.doi.org/10.1021/acs.biochem.6b01141DOI Listing
February 2017

Recombinant human dihydroxyacetonephosphate acyl-transferase characterization as an integral monotopic membrane protein.

Biochem Biophys Res Commun 2016 Dec 9;481(1-2):51-58. Epub 2016 Nov 9.

Department of Biology and Biotechnology, University of Pavia, V. Ferrata 9, 27100, Pavia, Italy. Electronic address:

Although the precise functions of ether phospholipids are still poorly understood, significant alterations in their physiological levels are associated either to inherited disorders or to aggressive metastatic cancer. The essential precursor, alkyl-dihydroxyacetone phosphate (DHAP), for all ether phospholipids species is synthetized in two consecutive reactions performed by two enzymes sitting on the inner side of the peroxisomal membrane. Here, we report the characterization of the recombinant human DHAP acyl-transferase, which performs the first step in alkyl-DHAP synthesis. By exploring several expression systems and designing a number of constructs, we were able to purify the enzyme in its active form and we found that it is tightly bound to the membrane through the N-terminal residues.
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http://dx.doi.org/10.1016/j.bbrc.2016.11.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5146282PMC
December 2016

α-Synuclein is a Novel Microtubule Dynamase.

Sci Rep 2016 09 15;6:33289. Epub 2016 Sep 15.

Dept. Biosciences, Università degli Studi di Milano, Milano, Italy.

α-Synuclein is a presynaptic protein associated to Parkinson's disease, which is unstructured when free in the cytoplasm and adopts α helical conformation when bound to vesicles. After decades of intense studies, α-Synuclein physiology is still difficult to clear up due to its interaction with multiple partners and its involvement in a pletora of neuronal functions. Here, we looked at the remarkably neglected interplay between α-Synuclein and microtubules, which potentially impacts on synaptic functionality. In order to identify the mechanisms underlying these actions, we investigated the interaction between purified α-Synuclein and tubulin. We demonstrated that α-Synuclein binds to microtubules and tubulin α2β2 tetramer; the latter interaction inducing the formation of helical segment(s) in the α-Synuclein polypeptide. This structural change seems to enable α-Synuclein to promote microtubule nucleation and to enhance microtubule growth rate and catastrophe frequency, both in vitro and in cell. We also showed that Parkinson's disease-linked α-Synuclein variants do not undergo tubulin-induced folding and cause tubulin aggregation rather than polymerization. Our data enable us to propose α-Synuclein as a novel, foldable, microtubule-dynamase, which influences microtubule organisation through its binding to tubulin and its regulating effects on microtubule nucleation and dynamics.
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http://dx.doi.org/10.1038/srep33289DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5024109PMC
September 2016

Tools for the rational design of bivalent microtubule-targeting drugs.

Biochem Biophys Res Commun 2016 10 7;479(1):48-53. Epub 2016 Sep 7.

Dipartimento di Bioscienze, Università degli Studi di Milano, via Celoria 26, 20133 Milano, Italy. Electronic address:

Microtubule (MT) dynamic behaviour is an attractive drug target for chemotherapy, whose regulation by MT-stabilizing and destabilizing agents has been fruitfully applied in treating several types of cancers. MT-stabilizing agents are also emerging as potential remedies for neurodegenerative conditions, such as Alzheimer's and Parkinson's disease, although single-target drugs are not expected to fully cure these complex pathologies. Drug combination often displays enhanced efficacy with respect to mono-therapies. In particular, MT-targeting bivalent compounds (MTBCs) represent a promising class of molecules; however, surprisingly, the majority of MTBCs reported so far exhibit equal if not less efficacy than their building monomers. In order to shed light on MTBCs poor performance, we characterised through a set of complementary approaches thiocolchine (TH) and two bivalent TH-homodimers as prototype molecules. First, the binding affinities of these three molecules were assessed, then we obtained the crystallographic structure of a tubulin-TH complex. The binding affinities were interpreted in light of structural data and of molecular dynamics simulations. Finally, their effects on MT cytoskeleton and cell survival were validated on HeLa cells. The ensemble of these data provides chemical and structural considerations on how a successful rational design of MTBCs should be conceived.
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http://dx.doi.org/10.1016/j.bbrc.2016.09.022DOI Listing
October 2016

Key Role of the Adenylate Moiety and Integrity of the Adenylate-Binding Site for the NAD(+)/H Binding to Mitochondrial Apoptosis-Inducing Factor.

Biochemistry 2015 Dec 16;54(47):6996-7009. Epub 2015 Nov 16.

Department of Biosciences, Università degli Studi di Milano , via Celoria 26, 20133 Milano, Italy.

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein with pro-life and pro-death activities, which plays critical roles in mitochondrial energy metabolism and caspase-independent apoptosis. Defects in AIF structure or expression can cause mitochondrial abnormalities leading to mitochondrial defects and neurodegeneration. The mechanism of AIF-induced apoptosis was extensively investigated, whereas the mitochondrial function of AIF is poorly understood. A unique feature of AIF is the ability to form a tight, air-stable charge-transfer (CT) complex upon reaction with NADH and to undergo a conformational switch leading to dimerization, proposed to be important for its vital and lethal functions. Although some aspects of interaction of AIF with NAD(+)/H have been analyzed, its precise mechanism is not fully understood. We investigated how the oxidized and photoreduced wild-type and G307A and -E variants of murine AIF associate with NAD(+)/H and nicotinamide mononucleotide (NMN(+)/H) to determine the role of the adenylate moiety in the binding process. Our results indicate that (i) the adenylate moiety of NAD(+)/H is crucial for the association with AIF and for the subsequent structural reorganization of the complex, but not for protein dimerization, (ii) FAD reduction rather than binding of NAD(+)/H to AIF initiates conformational rearrangement, and (iii) alteration of the adenylate-binding site by the G307E (equivalent to a pathological G308E mutation in human AIF) or G307A replacements decrease the affinity and association rate of NAD(+)/H, which, in turn, perturbs CT complex formation and protein dimerization but has no influence on the conformational switch in the regulatory peptide.
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http://dx.doi.org/10.1021/acs.biochem.5b00898DOI Listing
December 2015

Discovery of Inhibitors for the Ether Lipid-Generating Enzyme AGPS as Anti-Cancer Agents.

ACS Chem Biol 2015 Nov 4;10(11):2589-97. Epub 2015 Sep 4.

Department of Biology and Biotechnology, University of Pavia , via Ferrata 9, 27100 Pavia, Italy.

Dysregulated ether lipid metabolism is an important hallmark of cancer cells. Previous studies have reported that lowering ether lipid levels by genetic ablation of the ether lipid-generating enzyme alkyl-glycerone phosphate synthase (AGPS) lowers key structural and oncogenic ether lipid levels and alters fatty acid, glycerophospholipid, and eicosanoid metabolism to impair cancer pathogenicity, indicating that AGPS may be a potential therapeutic target for cancer. In this study, we have performed a small-molecule screen to identify candidate AGPS inhibitors. We have identified several lead AGPS inhibitors and have structurally characterized their interactions with the enzyme and show that these inhibitors bind to distinct portions of the active site. We further show that the lead AGPS inhibitor 1a selectively lowers ether lipid levels in several types of human cancer cells and impairs their cellular survival and migration. We provide here the first report of in situ-active pharmacological tools for inhibiting AGPS, which may provide chemical scaffolds for future AGPS inhibitor development for cancer therapy.
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http://dx.doi.org/10.1021/acschembio.5b00466DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4703096PMC
November 2015

An improved expression system for the VC1 ligand binding domain of the receptor for advanced glycation end products in Pichia pastoris.

Protein Expr Purif 2015 Oct 25;114:48-57. Epub 2015 Jun 25.

Department of Biosciences, Via Celoria 26, University of Milan, 20133 Milano, Italy. Electronic address:

The receptor for the advanced glycation end products (RAGE) is a type I transmembrane glycoprotein belonging to the immunoglobulin superfamily and binds a variety of unrelated ligands sharing a negative charge. Most ligands bind to the extracellular V or VC1 domains of the receptor. In this work, V and VC1 of human RAGE were produced in the methylotrophic yeast Pichia pastoris and directed to the secretory pathway. Fusions to a removable C-terminal His-tag evidenced proteolytic processing of the tag by extracellular proteases and also intracellular degradation of the N-terminal portion of V-His. Expression of untagged forms was attempted. While the V domain was retained intracellularly, VC1 was secreted into the medium and was functionally active in binding AGEs. The glycosylation state of VC1 was analyzed by mass spectrometry and peptide-N-glycosidase F digestion. Like RAGE isolated from mammalian sources, the degree of occupancy of the N-glycosylation sites was full at Asn25 and partial at Asn81 which was also subjected to non-enzymatic deamidation. A simple procedure for the purification to homogeneity of VC1 from the medium was developed. The folded state of the purified protein was assessed by thermal shift assays. Recombinant VC1 from P. pastoris showed a remarkably high thermal stability as compared to the protein expressed in bacteria. Our in vivo approach indicates that the V and C1 domains constitute a single folding unit. The stability and solubility of the yeast-secreted VC1 may be beneficial for future in vitro studies aimed to identify new ligands or inhibitors of RAGE.
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http://dx.doi.org/10.1016/j.pep.2015.06.012DOI Listing
October 2015

Centaurin-α₂ interacts with β-tubulin and stabilizes microtubules.

PLoS One 2012 20;7(12):e52867. Epub 2012 Dec 20.

Dipartimento di Biotecnologie Mediche e Medicina Traslazionale, Università degli Studi di Milano, Milano, Italy.

Centaurin-α₂ is a GTPase-activating protein for ARF (ARFGAP) showing a diffuse cytoplasmic localization capable to translocate to membrane, where it binds phosphatidylinositols. Taking into account that Centaurin-α₂ can localize in cytoplasm and that its cytoplasmatic function is not well defined, we searched for further interactors by yeast two-hybrid assay to investigate its biological function. We identified a further Centaurin-α₂ interacting protein, β-Tubulin, by yeast two-hybrid assay. The interaction, involving the C-terminal region of β-Tubulin, has been confirmed by coimmunoprecipitation experiments. After Centaurin-α₂ overexpression in HeLa cells and extraction of soluble (αβ dimers) and insoluble (microtubules) fractions of Tubulin, we observed that Centaurin-α₂ mainly interacts with the polymerized Tubulin fraction, besides colocalizing with microtubules (MTs) in cytoplasm accordingly. Even following the depolimerizing Tubulin treatments Centaurin-α₂ remains mainly associated to nocodazole- and cold-resistant MTs. We found an increase of MT stability in transfected HeLa cells, evaluating as marker of stability the level of MT acetylation. In vitro assays using purified Centaurin-α₂ and tubulin confirmed that Centaurin-α₂ promotes tubulin assembly and increases microtubule stability. The biological effect of Centaurin-α₂ overexpression, assessed through the detection of an increased number of mitotic HeLa cells with bipolar spindles and with the correct number of centrosomes in both dividing and not dividing cells, is consistent with the Centaurin-α₂ role on MT stabilization. Centaurin-α₂ interacts with β-Tubulin and it mainly associates to MTs, resistant to destabilizing agents, in vitro and in cell. We propose Centaurin-α₂ as a new microtubule-associated protein (MAP) increasing MT stability.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0052867PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3527619PMC
July 2013

Is renalase a novel player in catecholaminergic signaling? The mystery of the catalytic activity of an intriguing new flavoenzyme.

Curr Pharm Des 2013 ;19(14):2540-51

Dipartimento di Bioscienze, Universita degli Studi di Milano, via Celoria 26, 20133 Milano, Italy.

Renalase is a flavoprotein recently discovered in humans, preferentially expressed in the proximal tubules of the kidney and secreted in blood and urine. It is highly conserved in vertebrates, with homologs identified in eukaryotic and prokaryotic organisms. Several genetic, epidemiological, clinical and experimental studies show that renalase plays a role in the modulation of the functions of the cardiovascular system, being particularly active in decreasing the catecholaminergic tone, in lowering blood pressure and in exerting a protective action against myocardial ischemic damage. Deficient renalase synthesis might be the cause of the high occurrence of hypertension and adverse cardiac events in kidney disease patients. Very recently, recombinant human renalase has been structurally and functionally characterized in vitro. Results show that it belongs to the p-hydroxybenzoate hydroxylase structural family of flavoenzymes, contains non-covalently bound FAD with redox features suggestive of a dehydrogenase activity, and is not a catecholamine-degrading enzyme,either through oxidase or NAD(P)H-dependent monooxygenase reactions. The biochemical data now available will hopefully provide the basis for a systematic and rational quest toward the identification of the reaction catalyzed by renalase and of the molecular mechanism of its physiological action, which in turn are expected to favor the development of novel therapeutic tools for the treatment of kidney and cardiovascular diseases.
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http://dx.doi.org/10.2174/1381612811319140005DOI Listing
September 2013

Precursor of ether phospholipids is synthesized by a flavoenzyme through covalent catalysis.

Proc Natl Acad Sci U S A 2012 Nov 29;109(46):18791-6. Epub 2012 Oct 29.

Department of Biology and Biotechnology, University of Pavia, 27100 Pavia, Italy.

The precursor of the essential ether phospholipids is synthesized by a peroxisomal enzyme that uses a flavin cofactor to catalyze a reaction that does not alter the redox state of the substrates. The enzyme crystal structure reveals a V-shaped active site with a narrow constriction in front of the prosthetic group. Mutations causing inborn ether phospholipid deficiency, a very severe genetic disease, target residues that are part of the catalytic center. Biochemical analysis using substrate and flavin analogs, absorbance spectroscopy, mutagenesis, and mass spectrometry provide compelling evidence supporting an unusual mechanism of covalent catalysis. The flavin functions as a chemical trap that promotes exchange of an acyl with an alkyl group, generating the characteristic ether bond. Structural comparisons show that the covalent versus noncovalent mechanistic distinction in flavoenzyme catalysis and evolution relies on subtle factors rather than on gross modifications of the cofactor environment.
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http://dx.doi.org/10.1073/pnas.1215128109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3503197PMC
November 2012

A single tyrosine hydroxyl group almost entirely controls the NADPH specificity of Plasmodium falciparum ferredoxin-NADP+ reductase.

Biochemistry 2012 May 30;51(18):3819-26. Epub 2012 Apr 30.

Department of Biomolecular Sciences and Biotechnology, Università degli Studi di Milano, via Celoria 26, 20133 Milano, Italy.

Plasmodium falciparum ferredoxin-NADP(+) reductase (FNR) is a FAD-containing enzyme that, in addition to be a promising target of novel antimalarial drugs, represents an excellent model of plant-type FNRs. The cofactor specificity of FNRs depends on differences in both k(cat) and K(m) values for NADPH and NADH. Here, we report that deletion of the hydroxyl group of the conserved Y258 of P. falciparum FNR, which interacts with the 2'-phosphate group of NADPH, selectively decreased the k(cat) of the NADPH-dependent reaction by a factor of 2 to match that of the NADH-dependent one. Rapid-reaction kinetics, active-site titrations with NADP(+), and anaerobic photoreduction experiments indicated that this effect may be the consequence of destabilization of the catalytically competent conformation of bound NADPH. Moreover, because the Y258F replacement increased the K(m) for NADPH 4-fold and decreased that for NADH 3-fold, it led to a drop in the ability of the enzyme to discriminate between the coenzymes from 70- to just 1.5-fold. The impact of the Y258F change was not affected by the presence of the H286Q mutation, which is known to enhance the catalytic activity of the enzyme. Our data highlight the major role played by the Y258 hydroxyl group in determining the coenzyme specificity of P. falciparum FNR. From the general standpoint of engineering the kinetic properties of plant-type FNRs, although P. falciparum FNR is less strictly NADPH-dependent than its homologues, the almost complete abolishment of coenzyme selectivity reported here has never been accomplished before through a single mutation.
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http://dx.doi.org/10.1021/bi300078pDOI Listing
May 2012

FAD-binding site and NADP reactivity in human renalase: a new enzyme involved in blood pressure regulation.

J Mol Biol 2011 Aug 14;411(2):463-73. Epub 2011 Jun 14.

Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, via Celoria 26, 20133 Milan, Italy.

Renalase is a recently discovered flavoprotein that regulates blood pressure, regulates sodium and phosphate excretion, and displays cardioprotectant action through a mechanism that is barely understood to date. It has been proposed to act as a catecholamine-degrading enzyme, via either O(2)-dependent or NADH-dependent mechanisms. Here we report the renalase crystal structure at 2.5 Å resolution together with new data on its interaction with nicotinamide dinucleotides. Renalase adopts the p-hydroxybenzoate hydroxylase fold topology, comprising a Rossmann-fold-based flavin adenine dinucleotide (FAD)-binding domain and a putative substrate-binding domain, the latter of which contains a five-stranded anti-parallel β-sheet. A large cavity (228 Å(3)), facing the flavin ring, presumably represents the active site. Compared to monoamine oxidase or polyamine oxidase, the renalase active site is fully solvent exposed and lacks an 'aromatic cage' for binding the substrate amino group. Renalase has an extremely low diaphorase activity, displaying lower k(cat) but higher k(cat)/K(m) for NADH compared to NADPH. Moreover, its FAD prosthetic group becomes slowly reduced when it is incubated with NADPH under anaerobiosis, and binds NAD(+) or NADP(+) with K(d) values of ca 2 mM. The absence of a recognizable NADP-binding site in the protein structure and its poor affinity for, and poor reactivity towards, NADH and NADPH suggest that these are not physiological ligands of renalase. Although our study does not answer the question on the catalytic activity of renalase, it provides a firm framework for testing hypotheses on the molecular mechanism of its action.
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http://dx.doi.org/10.1016/j.jmb.2011.06.010DOI Listing
August 2011

Synthesis of human renalase1 in Escherichia coli and its purification as a FAD-containing holoprotein.

Protein Expr Purif 2010 Aug 17;72(2):244-53. Epub 2010 Mar 17.

Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, via Celoria 26, 20133 Milano, Italy.

Renalase is a protein ubiquitous in vertebrates, which has been proposed to modulate blood pressure and heart rate, and whose downregulation might result in hypertension. Despite its potential relevance for human health, the biochemical characterization of renalase is still lacking, possibly due to difficulties in obtaining it in recombinant form. By expressing two different gene constructs, we found that the major isoform of human renalase, renalase1, is mainly produced in Escherichia coli in inclusion bodies. However, by optimizing the expression conditions, significant amounts of soluble products were obtained. Both soluble renalase forms have been purified to homogeneity exploiting their N-terminal His-tag. Linking of the protein of interest to the SUMO protein did not improve solubility, but yielded untagged renalase1 after proteolytic processing of the fusion product. The two recombinant renalase forms displayed the same molecular properties. They bind equimolar amounts of FAD and appear to be correctly folded by various criteria. The procedures for the production and isolation of recombinant renalase1 here reported are expected to boost the much awaited biochemical studies on this remarkable protein.
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http://dx.doi.org/10.1016/j.pep.2010.03.008DOI Listing
August 2010

Antiplasmodial activity of quinones: roles of aziridinyl substituents and the inhibition of Plasmodium falciparum glutathione reductase.

Arch Biochem Biophys 2010 Feb 15;494(1):32-9. Epub 2009 Nov 15.

Museum National d'Histoire Naturelle, FRE 3206 CNRS, 61 rue Buffon, Paris Cedex 05, France.

Although quinones have been the subject of great interest as possible antimalarial agents, the mechanism of their antimalarial activity is poorly understood. Flavoenzyme electrontransferase-catalyzed redox cycling of quinones, and their inhibition of the antioxidant flavoenzyme glutathione reductase (GR, EC 1.8.1.7) have been proposed as possible mechanisms. Here, we have examined the activity of a number of quinones, including the novel antitumor agent RH1, against the malaria parasite Plasmodium falciparum strain FcB1 in vitro, their single-electron reduction rates by P. falciparum ferredoxin:NADP(+) reductase (PfFNR, EC 1.18.1.2), and their ability to inhibit P. falciparum GR. The multiparameter statistical analysis of our data implies, that the antiplasmodial activity of fully-substituted quinones (n=15) is relatively independent from their one-electron reduction potential (E(7)(1)). The presence of aziridinyl groups in quinone ring increased their antiplasmodial activity. Since aziridinyl-substituted quinones do not possess enhanced redox cycling activity towards PfFNR, we propose that they could act as as DNA-alkylating agents after their net two-electron reduction into aziridinyl-hydroquinones. We found that under the partial anaerobiosis, i.e., at the oxygen concentration below 40-50 microM, this reaction may be carried out by single-electron transferring flavoenzymes present in P. falciparum, like PfFNR. Another parameter increasing the antiplasmodial activity of fully-substituted quinones is an increase in their potency as P. falciparum GR inhibitors, which was revealed using multiparameter regression analysis. To our knowledge, this is the first quantitative demonstration of a link between the antiplasmodial activity of compounds and GR inhibition.
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http://dx.doi.org/10.1016/j.abb.2009.11.012DOI Listing
February 2010

Plasmodium falciparum ferredoxin-NADP+ reductase His286 plays a dual role in NADP(H) binding and catalysis.

Biochemistry 2009 Oct;48(40):9525-33

Dipartimento di Scienze Biomolecolari e Biotecnologie, Universita degli Studi di Milano, via Celoria 26, 20133 Milano, Italy.

The NADP-binding site of Plasmodium falciparum ferredoxin-NADP(+) reductase contains two basic residues, His286 and Lys249, conserved within the Plasmodium genus, but not in other plant-type homologues. Previous crystal studies indicated that His286 interacts with the adenine ring and with the 5'-phosphate of 2'-P-AMP, a ligand that mimics the adenylate moiety of NADP(H). Here we show that replacement of His286 with aliphatic residues results both in a decrease in the affinity of the enzyme for NADPH and in a decrease in k(cat), due to a lowered hydride-transfer rate. Unexpectedly, the mutation to Gln produces an enzyme more active than the wild-type one, whereas the change to Lys destabilizes the nicotinamide-isoalloxazine interaction, decreasing k(cat). On the basis of the crystal structure of selected mutants complexed with 2'-P-AMP, we conclude that the His286 side chain plays a dual role in catalysis both by providing binding energy for NADPH and by favoring the catalytically competent orientation of its nicotinamide ring. For the latter function, the H-bonding potential rather than the positively charged state of the His286 imidazole seems sufficient. Furthermore, we show that the Lys249Ala mutation decreases K(m)(NADPH) and K(d) for NADP(+) or 2'-P-AMP by a factor of 10. We propose that the Lys249 side chain participates in substrate recognition by interacting with the 2'-phosphate of NADP(H) and that this interaction was not observed in the crystal form of the enzyme-2'-P-AMP complex due to a conformational perturbation of the substrate-binding loop induced by dimerization.
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http://dx.doi.org/10.1021/bi9013209DOI Listing
October 2009

The ferredoxin-NADP+ reductase/ferredoxin electron transfer system of Plasmodium falciparum.

FEBS J 2009 Jul 11;276(14):3825-36. Epub 2009 Jun 11.

Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, Italy.

In the apicoplast of apicomplexan parasites, plastidic-type ferredoxin and ferredoxin-NADP(+) reductase (FNR) form a short electron transport chain that provides reducing power for the synthesis of isoprenoid precursors. These proteins are attractive targets for the development of novel drugs against diseases such as malaria, toxoplasmosis, and coccidiosis. We have obtained ferredoxin and FNR of both Toxoplasma gondii and Plasmodium falciparum in recombinant form, and recently we solved the crystal structure of the P. falciparum reductase. Here we report on the functional properties of the latter enzyme, which differ markedly from those of homologous FNRs. In the physiological reaction, P. falciparum FNR displays a k(cat) five-fold lower than those usually determined for plastidic-type FNRs. By rapid kinetics, we found that hydride transfer between NADPH and protein-bound FAD is slower in the P. falciparum enzyme. The redox properties of the enzyme were determined, and showed that the FAD semiquinone species is highly destabilized. We propose that these two features, i.e. slow hydride transfer and unstable FAD semiquinone, are responsible for the poor catalytic efficiency of the P. falciparum enzyme. Another unprecedented feature of the malarial parasite FNR is its ability to yield, under oxidizing conditions, an inactive dimeric form stabilized by an intermolecular disulfide bond. Here we show that the monomerdimer interconversion can be controlled by oxidizing and reducing agents that are possibly present within the apicoplast, such as H(2)O(2), glutathione, and lipoate. This finding suggests that modulation of the quaternary structure of P. falciparum FNR might represent a regulatory mechanism, although this needs to be verified in vivo.
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http://dx.doi.org/10.1111/j.1742-4658.2009.07100.xDOI Listing
July 2009

Structural and functional diversity of ferredoxin-NADP(+) reductases.

Arch Biochem Biophys 2008 Jun 16;474(2):283-91. Epub 2008 Feb 16.

Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, via Celoria 26, 20133 Milano, Italy.

Although all ferredoxin-NADP(+) reductases (FNRs) catalyze the same reaction, i.e. the transfer of reducing equivalents between NADP(H) and ferredoxin, they belong to two unrelated families of proteins: the plant-type and the glutathione reductase-type of FNRs. Aim of this review is to provide a general classification scheme for these enzymes, to be used as a framework for the comparison of their properties. Furthermore, we report on some recent findings, which significantly increased the understanding of the structure-function relationships of FNRs, i.e. the ability of adrenodoxin reductase and its homologs to catalyze the oxidation of NADP(+) to its 4-oxo derivative, and the properties of plant-type FNRs from non-photosynthetic organisms. Plant-type FNRs from bacteria and Apicomplexan parasites provide examples of novel ways of FAD- and NADP(H)-binding. The recent characterization of an FNR from Plasmodium falciparum brings these enzymes into the field of drug design.
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http://dx.doi.org/10.1016/j.abb.2008.02.014DOI Listing
June 2008

Effect of salt and pH on the reductive half-reaction of Mycobacterium tuberculosis FprA with NADPH.

Biochemistry 2008 Mar 23;47(11):3418-25. Epub 2008 Feb 23.

Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, via Celoria 26, 20133 Milano, Italy.

Despite a number of studies, the formation of the Michaelis complexes between ferredoxin-NADP (+) reductases and NADP(H) eluded detailed investigations by rapid kinetic techniques because of their high formation rates. Moreover, the reversible nature of the reaction of hydride ion transfer between these enzymes and NADPH prevented the obtainment of reliable estimates of the rate constant of the hydride transfer step. Here we show that by working at a high salt concentration, the mechanism of the reaction with NADPH of FprA, a Mycobacterium tuberculosis homologue of adrenodoxin reductase, is greatly simplified, making it amenable to investigation by rapid reaction techniques. The approach presented herein allowed for the first time the observation of the formation of the Michaelis complex between an adrenodoxin reductase-like enzyme and NADPH, and the determination of the related rate constants for association and dissociation. Furthermore, the rate constant for the reaction of hydride ion transfer between NADPH and FAD could be unambiguously assessed. It is proposed that the approach described should be applicable to other ferredoxin reductase enzymes, providing a valuable experimental tool for the study of their kinetic properties.
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http://dx.doi.org/10.1021/bi702250hDOI Listing
March 2008

Enzymatic oxidation of NADP+ to its 4-oxo derivative is a side-reaction displayed only by the adrenodoxin reductase type of ferredoxin-NADP+ reductases.

FEBS J 2007 Aug 16;274(15):3998-4007. Epub 2007 Jul 16.

Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, Italy.

We have previously shown that Mycobacterium tuberculosis FprA, an NADPH-ferredoxin reductase homologous to mammalian adrenodoxin reductase, promotes the oxidation of NADP(+) to its 4-oxo derivative 3-carboxamide-4-pyridone adenine dinucleotide phosphate [Bossi RT, Aliverti A, Raimondi D, Fischer F, Zanetti G, Ferrari D, Tahallah N, Maier CS, Heck AJ, Rizzi M et al. (2002) Biochemistry41, 8807-8818]. Here, we provide a detailed study of this unusual enzyme reaction, showing that it occurs at a very slow rate (0.14 h(-1)), requires the participation of the enzyme-bound FAD, and is regiospecific in affecting only the C4 of the NADP nicotinamide ring. By protein engineering, we excluded the involvement in catalysis of residues Glu214 and His57, previously suggested to be implicated on the basis of their localization in the three-dimensional structure of the enzyme. Our results substantiate a catalytic mechanism for 3-carboxamide-4-pyridone adenine dinucleotide phosphate formation in which the initial and rate-determining step is the nucleophilic attack of the nicotinamide moiety by an active site water molecule. Whereas plant-type ferredoxin reductases were unable to oxidize NADP(+), the mammalian adrenodoxin reductase also catalyzed this unusual reaction. Thus, the 3-carboxamide-4-pyridone adenine dinucleotide phosphate formation reaction seems to be a peculiar feature of the mitochondrial type of ferredoxin reductases, possibly reflecting conserved properties of their active sites. Furthermore, we showed that 3-carboxamide-4-pyridone adenine dinucleotide phosphate is good ligand and a competitive inhibitor of various dehydrogenases, making this nucleotide analog a useful tool for the characterization of the cosubstrate-binding site of NADPH-dependent enzymes.
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http://dx.doi.org/10.1111/j.1742-4658.2007.05934.xDOI Listing
August 2007

The crystal structure of FdxA, a 7Fe ferredoxin from Mycobacterium smegmatis.

Biochem Biophys Res Commun 2007 Aug 12;360(1):97-102. Epub 2007 Jun 12.

Department of Biomolecular Sciences and Biotechnology, University of Milano, Via Celoria 26, 20133 Milano, Italy.

Mycobacterium smegmatis ferredoxin FdxA, which has an orthologue ferredoxin in Mycobacterium tuberculosis, FdxC, contains both one [3Fe-4S] and one [4Fe-4S] cluster. M. smegmatis FdxA has been shown to be a preferred ferredoxin substrate of FprA [F. Fischer, D. Raimondi, A. Aliverti, G. Zanetti, Mycobacterium tuberculosis FprA, a novel bacterial NADPH-ferredoxin reductase, Eur. J. Biochem. 269 (2002) 3005-3013], an adrenodoxin reductase-like flavoprotein of M. tuberculosis, suggesting that M. tuberculosis FdxC could be the physiological partner of the enzyme in providing reducing power to the cytochromes P450. We report here the crystal structure of FdxA at 1.6A resolution (R(factor) 16.5%, R(free) 20.2%). Besides providing an insight on protein architecture for this 106-residue ferredoxin, our crystallographic investigation highlights lability of the [4Fe-4S] center, which is shown to loose a Fe atom during crystal growth. Due to their high similarity (87% sequence identity), the structure here reported can be considered a valuable model for M. tuberculosis FdxC, thus representing a step forward in the study of the complex mycobacterial redox pathways.
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http://dx.doi.org/10.1016/j.bbrc.2007.06.013DOI Listing
August 2007

The crucial step in ether phospholipid biosynthesis: structural basis of a noncanonical reaction associated with a peroxisomal disorder.

Structure 2007 Jun;15(6):683-92

Dipartimento di Genetica e Microbiologia, Università di Pavia, via Ferrata 1, 27100 Pavia, Italy.

Ether phospholipids are essential constituents of eukaryotic cell membranes. Rhizomelic chondrodysplasia punctata type 3 is a severe peroxisomal disorder caused by inborn deficiency of alkyldihydroxyacetonephosphate synthase (ADPS). The enzyme carries out the most characteristic step in ether phospholipid biosynthesis: formation of the ether bond. The crystal structure of ADPS from Dictyostelium discoideum shows a fatty-alcohol molecule bound in a narrow hydrophobic tunnel, specific for aliphatic chains of 16 carbons. Access to the tunnel is controlled by a flexible loop and a gating helix at the protein-membrane interface. Structural and mutagenesis investigations identify a cluster of hydrophilic catalytic residues, including an essential tyrosine, possibly involved in substrate proton abstraction, and the arginine that is mutated in ADPS-deficient patients. We propose that ether bond formation might be orchestrated through a covalent imine intermediate with the flavin, accounting for the noncanonical employment of a flavin cofactor in a nonredox reaction.
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http://dx.doi.org/10.1016/j.str.2007.04.009DOI Listing
June 2007